Therapeutic cell washing, concentration, and separation utilizing acoustophoresis

ABSTRACT

Multi-stage acoustophoretic devices for continuously separating a second fluid or a particulate from a host fluid are disclosed. Methods of operating the multi-stage acoustophoretic devices are also disclosed. The systems may include multiple acoustophoretic devices fluidly connected to one another in series, each acoustophoretic device comprising a flow chamber, an ultrasonic transducer capable of creating a multi-dimensional acoustic standing wave, and a reflector. The systems can further include pumps and flowmeters.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/942,427, filed on Mar. 30, 2018, which is a continuation-in-part ofU.S. patent application Ser. No. 15/586,116, filed on May 3, 2017, nowU.S. Pat. No. 10,640,760, which claims priority to U.S. ProvisionalPatent Application Ser. No. 62/330,947, filed on May 3, 2016, and toU.S. Provisional Patent Application Ser. No. 62/359,182, filed on Jul.6, 2016, and to U.S. Provisional Patent Application Ser. No. 62/374,910,filed on Aug. 15, 2016. U.S. patent application Ser. No. 15/942,427,filed on Mar. 30, 2018 also claims the benefit of U.S. ProvisionalPatent Application Ser. No. 62/479,309, filed on Mar. 30, 2017. Thedisclosures of these applications are hereby fully incorporated hereinby reference in their entirety.

BACKGROUND

Concentrating therapeutic cells and transferring them from one solutioninto another (usually referred to as washing) are two processes involvedat multiple stages of production and use of the cells. The washing andseparation of materials in cellular processing is an important part ofthe overall efficacy of the cell therapy of choice. In particular,therapeutic cells may originally be suspended in a growth serum or inpreservative materials like dimethyl sulfoxide (DMSO). Separating thecells from these fluids so the cells can be further processed isimportant in the overall therapeutic process of using such cellularmaterials. In one example, the cells are typically recovered from abioreactor, concentrated, and transferred from culture media into anelectroporation buffer prior to transduction, such as in manufacturingCAR-T cells. After expansion of cells at the final manufacturing step,they are concentrated and transferred into an appropriate solventdepending on the desired application.

Therapeutic cells are stored in specialized media to prolong theviability of these cells either through refrigeration and or freezingprocesses. Such specialized media may not be compatible when thetherapeutic cells are introduced into a patient. It may thus be helpfulto both wash and concentrate the therapeutic cells in a buffer or washmedia that is biocompatible with both the therapeutic cells and with thepatient. These washing and concentration processes conventionallyinvolve the use of centrifugation and physical filtration. The washingstep may be repeated a number of times. For example, the specializedmedia (which can be pyrogenic or otherwise harmful) may be fully removedwith multiple wash steps, and the cells may be suspended in a new bufferor wash solution. During this washing process, many of the cells aredegraded or destroyed through the centrifugation and physical filtrationprocesses. Moreover, the filtration process can be rather inefficientand may entail a non-sterile intrusion into the environment for batchprocessing, whereby the cell culture is exposed to possible pathogens oroutside cellular influences that would be harmful to the target cellculture. Further yet, with these physical filtration processes,biological waste is generated through the use of multiple physicalfilters which may incur additional steps for proper disposal. The costand timeliness of this process is also not conducive to a fast orlow-cost process of preparing the cells for introduction to the patient.

BRIEF SUMMARY

The present disclosure provides methods and systems for replacing oraugmenting conventional centrifugation and physical filtration processesalong with the multiple washing steps with a simpler, lower cost, andmore friendly process for particles such as therapeutic cells. Themethods/processes can be performed in a sterile environment and in acontinuous form.

Disclosed herein are methods of washing particles, which may be cells.In some example methods, an initial mixture of a first media and theparticles is fed to a flow chamber of an acoustophoretic device. Thefirst media may contain preservatives such as dimethyl sulfoxide (DMSO)which are undesirable for future applications/uses of the particles. Theacoustophoretic device has at least one ultrasonic transducer thatincludes a piezoelectric material and is configured to be driven tocreate a multi-dimensional acoustic standing wave in the flow chamber.At least a portion of the particles are trapped in the multi-dimensionalacoustic standing wave. A second media is flowed through the flowchamber to wash out the first media while the particles are retained inthe multidimensional acoustic standing wave. The particles may thusexperience a media exchange, where the first media is exchanged for thesecond media.

In some examples, the volume of the second media used to perform thewash process may be equivalent to a volume of the flow chamber. In someexamples, the volume of the second media used to perform the washprocess may be multiples of or portions of the volume of the flowchamber. The second media can be a biocompatible wash or a buffersolution.

The particles may be cells. The cells may be Chinese hamster ovary (CHO)cells, NS0 hybridoma cells, baby hamster kidney (BHK) cells, humancells, regulatory T-cells, Jurkat T-cells, CAR-T cells, B cells, or NKcells, peripheral blood mononuclear cells (PBMCs), algae, plant cells,bacteria, or viruses. The cells may be attached to microcarriers.

Sometimes, the piezoelectric material of the at least one ultrasonictransducer is in the form of a piezoelectric array formed from aplurality of piezoelectric elements. Each piezoelectric element can bephysically separated from surrounding piezoelectric elements by apotting material. The piezoelectric array can be present on a singlecrystal, with one or more channels separating the piezoelectric elementsfrom each other. Each piezoelectric element can be individuallyconnected to its own pair of electrodes. The piezoelectric elements canbe operated in phase with each other, or operated out of phase with eachother. The acoustophoretic device may further comprise a cooling unitfor cooling the at least one ultrasonic transducer.

In various embodiments, the initial mixture may have a density of about0.5 million particles/mL to about 5 million particles/mL. Theconcentrated volume can be 25 to about 50 times less than a volume ofthe initial mixture. The concentrated volume may have a particle densityof 25 to about 50 times greater than a particle density of the initialmixture.

Also disclosed in various embodiments are methods of recovering greaterthan 90% of cells from a cell culture. An initial mixture of a firstmedia and the cell culture is fed through a flow chamber of anacoustophoretic device, the acoustophoretic device comprising at leastone ultrasonic transducer including a piezoelectric material that isconfigured to be driven to create a multi-dimensional acoustic standingwave in the flow chamber. The at least one ultrasonic transducer isdriven to create a multi-dimensional acoustic standing wave in the flowchamber, and thus to concentrate the cell culture within the acousticstanding wave. The initial mixture has an initial cell density of about0.5 million cells/mL to about 5 million cells/mL, and the concentratedcell culture has a cell density at least 25 times greater than theinitial cell density.

In some embodiments, the concentrated cell culture has a cell density of25 to about 50 times greater than the initial cell density. In otherembodiments, a volume of the concentrated cell culture is 25 to about 50times less than a volume of the initial mixture. The concentrated cellculture can be obtained in about 35 minutes or less.

Also disclosed are acoustophoretic devices, comprising: a flow chamberhaving a fluid inlet, a first outlet, and a second outlet; at least oneultrasonic transducer proximate a first wall of the flow chamber, atleast one ultrasonic transducer including a piezoelectric material thatis adapted to be driven to create a multi-dimensional acoustic standingwave; a reflector on a second wall of the flow chamber opposite the atleast one ultrasonic transducer; and a thermoelectric generator locatedbetween the at least one ultrasonic transducer and the first wall of theflow chamber.

The acoustophoretic device may have a concentrated volume of about 25 mLto about 75 mL. The acoustophoretic device may have a cell capacity ofabout 4 billion to about 40 billion cells. Various lines can connect theacoustophoretic device to containers that provide or receive variousmaterials to/from the acoustophoretic device.

These and other non-limiting characteristics are more particularlydescribed below.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following is a brief description of the drawings, which arepresented for the purposes of illustrating the example embodimentsdisclosed herein and not for the purposes of limiting the same.

FIG. 1 illustrates an example acoustophoresis process using a transducerand reflector to create an acoustic standing wave for trapping particlesand separating them from a fluid by enhanced gravitational settling.

FIG. 2 illustrates an example cell concentration and washing process(“diafiltration”) according to the present disclosure usingacoustophoresis.

FIG. 3 illustrates another example cell concentration and washingprocess (push through) according to the present disclosure usingacoustophoresis.

FIG. 4 shows six photographs that, from left to right and top to bottom,show the progression of cells being trapped in an acoustophoretic devicebefore a second media mixture (dyed blue) is flowed into the device andgradually replaces the first media (dyed red).

FIG. 5 is a perspective view of an example acoustophoretic deviceaccording to the present disclosure.

FIG. 6 is a cross-sectional illustration of the acoustophoretic deviceof FIG. 5.

FIG. 7 is a graph showing the performance of the acoustophoretic deviceof FIG. 5. The x-axis is elapsed time (minutes) and runs from 0 to 40 inincrements of 5. The left-side y-axis is permeate density reduction (%)and runs from 0 to 100 in increments of 10. The right-side y-axis ispermeate cell density (×10⁶ cells/m L) and runs from 0.00 to 2.00 inincrements of 0.20. The uppermost solid line represents permeatereduction density (%). The lowermost solid line represents permeate celldensity. The middle line running substantially horizontally across thepage represents feed cell density for reference purposes.

FIG. 8 is a conventional single-piece monolithic piezoelectric materialused in an ultrasonic transducer.

FIG. 9 is an example rectangular piezoelectric array having 16piezoelectric elements used in the transducers of the presentdisclosure.

FIG. 10 is another example rectangular piezoelectric array having 25piezoelectric elements used in the transducers of the presentdisclosure.

FIG. 11 is a diagram illustrating a piezoelectric material having 16piezoelectric elements operated in out-of-phase modes. Dark elementsindicate a 0° phase angle and light elements indicate a 180° phaseangle.

FIG. 12 illustrates a kerfed piezoelectric material (top) versus atransducer array that has piezoelectric elements joined together by apotting material (bottom).

FIG. 13 is a graph showing the performance of an acoustophoretic deviceaccording to the present disclosure having a 16-element piezoelectricarray, with the elements operated in-phase with one another. The x-axisis elapsed time (minutes) and runs from 0 to 60 in increments of 10. Theleft-side y-axis is permeate density reduction (%) and runs from 0 to100 in increments of 10. The right-side y-axis is permeate cell density(×10⁶ cells/mL) and runs from 0.00 to 2.50 in increments of 0.50. Theuppermost solid line represents permeate reduction density (%). Thelowermost solid line represents permeate cell density. The middle linerunning substantially horizontally across the page represents feed celldensity for reference purposes.

FIG. 14 is a graph showing the T-cell concentration performance of anacoustophoretic process according to the present disclosure with a lowcell density culture. The x-axis is elapsed time (minutes) and runs from0 to 25 in increments of 5. The left-side y-axis is percent reduction(%) and runs from 0 to 100 in increments of 10. The right-side y-axis iscell density (×10⁶ cells/mL) and runs from 0.00 to 1.60 in increments of0.20. The upper solid line represents permeate reduction (%). The lowersolid line represents permeate cell density. The dashed line representsfeed cell density for reference purposes.

FIG. 15 is a graph showing the percent density reduction (PDR)dependency on concentration and flow rate for an acoustophoretic processaccording to the present disclosure. The x-axis is time (minutes) andruns from 0 to 40 in increments of 5. The y-axis is permeate densityreduction (%) and runs from 0 to 100 in increments of 10. The linehaving circle-shaped data points represents a mixture having an initialcell concentration of 5×10⁶ cells/mL. The line having x-shaped datapoints represents a mixture having an initial cell concentration of3×10⁶ cells/mL. The line having triangle-shaped data points represents amixture having an initial cell concentration of 1×10⁶ cells/mL at a flowrate of 20 mL/minute. The line having diamond-shaped data pointsrepresents a mixture having an initial cell concentration of 1×10⁶cells/mL at a flow rate of 10 mL/minute.

FIG. 16 is a graph showing the T-cell performance for an acoustophoreticprocess according to the present disclosure with a high cell densityculture. The x-axis is elapsed time (minutes) and runs from 0 to 25 inincrements of 5. The left-side y-axis is percent reduction (%) and runsfrom 0 to 100 in increments of 10. The right-side y-axis is cell density(×10⁶ cells/m L) and runs from 0.00 to 3.00 in increments of 0.50. Theupper solid line represents permeate density reduction (%). The lowersolid line represents permeate cell density. The dashed line representsfeed cell density for reference purposes.

FIG. 17A is a perspective view of an example acoustophoretic deviceaccording to the present disclosure including a cooling unit for coolingthe transducer. FIG. 17B is an exploded view of the device of FIG. 17A.

FIG. 18 is a graph showing the temperature profile of an acoustophoreticdevice without active cooling. The x-axis is elapsed time (minutes) andruns from 0.00 to 20.00 in increments of 2.00. The y-axis is temperature(° C.) and runs from 17.00 to 33.00 in increments of 2.00. The lowermostline along the right side of the graph represents the feed temperature(° C.). The uppermost line along the right side of the graph representsthe core temperature (° C.). The middle line along the right side of thegraph represents the permeate temperature (° C.).

FIG. 19 is a graph showing the temperature profile of an acoustophoreticdevice with active cooling of the transducer. The x-axis is elapsed time(minutes) and runs from 0.00 to 20.00 in increments of 2.00. The y-axisis temperature (° C.) and runs from 17.00 to 33.00 in increments of2.00. The lowermost line along the right side of the graph representsthe feed temperature (° C.). The uppermost line along the right side ofthe graph represents the core temperature (° C.). The middle line alongthe right side of the graph represents the permeate temperature (° C.).

FIG. 20 illustrates a process for concentrating, washing, and/orseparating microcarriers and cells according to the present disclosure.The leftmost portion represents a first step of receiving complexes ofmicrocarriers and cells surrounded by a bioreactor serum from abioreactor and concentrating the microcarrier/cell complexes in anacoustophoretic device according to the present disclosure. The middleportion represents a second step of washing the concentratedmicrocarriers with attached cells to remove the bioreactor serum. Therightmost portion represents a third step of trypsinizing, ordisassociating, the microcarriers and cells and a fourth step ofseparating the microcarriers from the cells. The bottom portionrepresents a final wash and concentrate step that can be employed asdesired.

FIG. 21 shows media exchange in an acoustophoretic device according tothe present disclosure. The “Concentrate” photograph shows theconcentrate (e.g., concentrated microcarriers with attached T cells)surrounded by a first media (dyed red). The “Wash Pass 1” photographshows the microcarriers with attached T cells after a first wash passusing a second media (dyed blue). The “Wash Pass 2” photograph shows themicrocarriers with attached T cells after a second wash pass. Therightmost “Wash Pass 3” photograph shows the microcarriers with attachedT cells after a third wash pass, and is almost completely blue.

FIG. 22 shows microscopic images of the media exchange shown in FIG. 21.

FIG. 22 shows a microscopic image of the microcarriers with T attachedcells in the feed and during the three wash passes, and theconcentration of separated microcarriers and T cells in the permeate.

FIG. 23 shows the concentration of T cells in the acoustophoretic devicebefore acoustophoresis (top row of photographs) and after oneacoustophoresis pass (bottom row of photographs).

FIG. 24 shows the concentration of microcarriers with attached T cellsin the feed into the acoustophoretic device (top row of photographs) andthe concentration of separated microcarriers and T cells in the permeatedrawn out of the acoustophoretic device (bottom row of photographs). Thedark circular items indicate microcarriers, and the lighter areaindicates T cells.

FIG. 25 shows microscopic images of the concentration of microcarrierswith attached T cells in the feed and the concentration of separatedmicrocarriers and T cells in the permeate.

FIG. 26 is a schematic of an example acoustophoretic system according tothe present disclosure showing the flow path of the feed materialthrough the system.

FIG. 27 is a schematic of the example acoustophoretic system of FIG. 28showing the flow path of the wash material through the system.

FIG. 28 is a schematic of the example acoustophoretic system of FIG. 28showing draining of the system.

FIG. 29 is a two-axis graph showing the results of trial A. Theleft-hand y-axis is the percent reduction of cells in the permeate, andruns from 0 to 100% at intervals of 20%. The right-hand y-axis is thecell density of the permeate in units of million cells/m L, and runsfrom 0 to 1.00 at intervals of 0.20. The x-axis is elapsed time inminutes, and runs from 0 to 33 minutes at intervals of 3. The dottedline indicates the initial cell density, which was 0.98 millioncells/mL.

FIG. 30 is a two-axis graph showing the results of trial B. Theleft-hand y-axis is the percent reduction of cells in the permeate, andruns from 0 to 100% at intervals of 20%. The right-hand y-axis is thecell density of the permeate in units of million cells/m L, and runsfrom 0 to 1.00 at intervals of 0.20. The x-axis is elapsed time inminutes, and runs from 0 to 33 minutes at intervals of 3. The dottedline indicates the initial cell density, which was 0.85 millioncells/mL.

FIG. 31 is a two-axis graph showing the results of trial C. Theleft-hand y-axis is the percent reduction of cells, and runs from 0 to100% at intervals of 20%. The right-hand y-axis is the cell density inunits of million cells/mL, and runs from 0 to 4.00 at intervals of 1.00.The x-axis is elapsed time in minutes, and runs from 0 to 30 minutes atintervals of 3. The dotted line indicates the initial cell density,which was 4.08 million cells/mL.

FIG. 32 is a graph showing the absorbance at different wavelengths forsix different samples. Those samples are: 100% wash media (100 W-0 G),50% wash media and 50% growth media (50 W-50 G), 100% growth media (0W-100 G), first volume of the wash (1 Volume), second volume of the wash(2 Volume), and third volume of the wash (3 Volume). The y-axis isabsorbance, and runs from 0 to 1 at intervals of 0.1. The x-axis iswavelength, and runs from 540 nm to 640 nm at intervals of 50 nm.

DETAILED DESCRIPTION

The present disclosure may be understood more readily by reference tothe following detailed description of desired embodiments and theexamples included therein. In the following specification and the claimswhich follow, reference will be made to a number of terms which shall bedefined to have the following meanings.

Although specific terms are used in the following description for thesake of clarity, these terms are intended to refer only to theparticular structure of the embodiments selected for illustration in thedrawings, and are not intended to define or limit the scope of thedisclosure. In the drawings and the following description below, it isto be understood that like numeric designations refer to components oflike function. Furthermore, it should be understood that the drawingsare not to scale.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise.

As used in the specification and in the claims, the term “comprising”may include the embodiments “consisting of” and “consisting essentiallyof.” The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that require thepresence of the named components/steps and permit the presence of othercomponents/steps. However, such description should be construed as alsodescribing compositions or processes as “consisting of” and “consistingessentially of” the enumerated components/steps, which allows thepresence of only the named components/steps, along with any impuritiesthat might result therefrom, and excludes other components/steps.

Numerical values should be understood to include numerical values whichare the same when reduced to the same number of significant figures andnumerical values which differ from the stated value by less than theexperimental error of conventional measurement technique of the typedescribed in the present application to determine the value.

All ranges disclosed herein are inclusive of the recited endpoint andindependently combinable (for example, the range of “from 2 grams to 10grams” is inclusive of the endpoints, 2 grams and 10 grams, and all theintermediate values).

A value modified by a term or terms, such as “about” and“substantially,” may not be limited to the precise value specified. Theapproximating language may correspond to the precision of an instrumentfor measuring the value. The modifier “about” should also be consideredas disclosing the range defined by the absolute values of the twoendpoints. For example, the expression “from about 2 to about 4” alsodiscloses the range “from 2 to 4.”

It should be noted that many of the terms used herein are relativeterms. For example, the terms “upper” and “lower” are relative to eachother in location, e.g. an upper component is located at a higherelevation than a lower component in a given orientation, but these termscan change if the device is flipped. The terms “inlet” and “outlet” arerelative to a fluid flowing through them with respect to a givenstructure, e.g. a fluid flows through the inlet into the structure andflows through the outlet out of the structure. The terms “upstream” and“downstream” are relative to the direction in which a fluid flowsthrough various components, e.g. the flow fluids through an upstreamcomponent prior to flowing through the downstream component. It shouldbe noted that in a loop, a first component can be described as beingboth upstream of and downstream of a second component.

The terms “horizontal” and “vertical” are used to indicate directionrelative to an absolute reference, e.g. ground level. The terms“upwards” and “downwards” are also relative to an absolute reference; anupwards flow is always against the gravity of the earth.

The present application refers to “the same order of magnitude.” Twonumbers are of the same order of magnitude if the quotient of the largernumber divided by the smaller number is a value of at least 1 and lessthan 10.

The acoustophoretic technology of the present disclosure employsacoustic standing waves to concentrate, wash, and/or separate materials(such as particles or a secondary fluid) in a primary or host fluid. Inparticular, as shown in the upper left image (A) of FIG. 1, anultrasonic transducer T creates an acoustic wave in the fluid, whichinteracts with a reflector R positioned across from the ultrasonictransducer to create an acoustic standing wave. Although a reflector Ris illustrated in FIG. 1, another transducer may be used to reflectand/or generate acoustic energy to form the acoustic standing wave.

As shown in the upper right image (B) of FIG. 1, as the host fluid andmaterial entrained in the host fluid flows upwards through the acousticstanding wave, the acoustic standing wave(s) traps (retains or holds)the material (e.g., secondary phase materials, including fluids and/orparticles). The scattering of the acoustic field off the materialresults in a three-dimensional acoustic radiation force, which acts as athree-dimensional trapping field.

The three-dimensional acoustic radiation force generated in conjunctionwith an ultrasonic standing wave is referred to in the presentdisclosure as a three-dimensional or multi-dimensional standing wave.The acoustic radiation force is proportional to the particle volume(e.g. the cube of the radius) of the material when the particle is smallrelative to the wavelength. The acoustic radiation force is proportionalto frequency and the acoustic contrast factor. The acoustic radiationforce scales with acoustic energy (e.g. the square of the acousticpressure amplitude). For harmonic excitation, the sinusoidal spatialvariation of the force drives the particles to the stable positionswithin the standing waves. When the acoustic radiation force exerted onthe particles is stronger than the combined effect of fluid drag forceand buoyancy and gravitational force, the particle can be trapped withinthe acoustic standing wave field, as shown in the upper right image (B)of FIG. 1.

As can be seen in the lower left image (C) of FIG. 1, this trappingresults in coalescing, clumping, aggregating, agglomerating, and/orclustering of the trapped particles. Additionally, secondaryinter-particle forces, such as Bjerkness forces, aid in particleagglomeration.

As the particles continue to coalesce, clump, aggregate, agglomerate,and/or cluster the particles can grow to a certain size at whichgravitational forces on the particle cluster overcome the acousticradiation force. At such size, the particle cluster can fall out of theacoustic standing wave, as shown in the lower right image (D) of FIG. 1.

Desirably, the ultrasonic transducer(s) generate a three-dimensional ormulti-dimensional acoustic standing wave in the fluid that exerts alateral force on the suspended particles to accompany the axial force soas to increase the particle trapping capabilities of the standing wave.A planar or one-dimensional acoustic standing wave may provide acousticforces in the axial or wave propagation direction. The lateral force inplanar or one-dimensional acoustic wave generation may be two orders ofmagnitude smaller than the axial force. The multi-dimensional acousticstanding wave may provide a lateral force that is significantly greaterthan that of the planar acoustic standing wave. For example, the lateralforce may be of the same order of magnitude as the axial force in themulti-dimensional acoustic standing wave.

The acoustic standing waves of the present disclosure can be used totrap particles (e.g. therapeutic cells such as T cells, B cells, NKcells) suspended in a first media in the standing wave. The first mediacan then be replaced with a second media (e.g., a biocompatible wash orbuffer solution). Put another way, the host fluid of the particles canbe replaced. Prior to replacing the first media with the second media,acoustophoresis can be used to perform a diafiltration process, as shownin FIG. 2.

In FIG. 2, starting with an initial mixture that has a low cell densityof, for example, less than 1×10⁶ cells/mL, acoustophoresis can be usedto reduce the volume of the initial mixture, for example by at least10×, including 20× and up to 100× or more. The cell concentration may beincreased by at least 10×, including 20× and up to 100× or more. Thisinitial reduction process is the first volume reduction step (A). Next,the second media (e.g., a biocompatible wash or buffer solution) can beintroduced to at least partially displace the first media, as indicatedin step (B). Next, the new mixture of the cells and second media can besubjected to an acoustophoretic volume reduction step (C). This seriesof operations is referred to as a “diafiltration” process.

FIG. 3 illustrates a single-step, push-through process in whichparticles/cells are trapped in the acoustic standing wave and held inthe acoustophoretic device. The second media (e.g., a biocompatible washor buffer solution) is then flowed into the acoustophoretic device toeffectively “wash out” the first media. With the push-through process,more than 90%, including up to 99% or more, of the first media can beremoved from the particles/cells. The push-through process can beemployed as a continuous, single-use process that uses less buffersolution and less time than the diafiltration process of FIG. 2.

FIG. 4 shows six photographs that, from left to right and top to bottom,show the progression of cells being trapped in an acoustophoretic devicebefore a second media mixture (dyed blue) is flowed into the device andgradually replaces the first media (dyed red). In FIG. 4, a 150 mL feedvolume was used with 80 mL of electroporation media wash for the secondmedia. The concentrate was drawn off at a flow rate of 10 mL/minute. Ascan be seen in these pictures, over time the first media is replacedwith the second media.

With reference now to FIG. 5 and FIG. 6, a first example embodiment ofan acoustophoretic device 100 for separation of particles/cells fromfluid is depicted. The acoustophoretic device 100 includes a flowchamber 110 having at least one inlet and at least one outlet. In thisembodiment, the flow chamber 110 includes inlet 112, permeate outlet114, concentrate outlet 116, an ultrasonic transducer 120, and areflector 130. The inlet 112 can, in certain embodiments, serve the dualfunction of introducing the cells surrounded by the first media into theflow chamber 110 in addition to introducing the second media into theflow chamber 110. Alternatively, separate inlets can be used forintroducing the first and second media.

The flow chamber 110 is the region of the device 100 through which isflowed the cells surrounded by the first media. In this embodiment, theflow chamber 110 is defined by inlet 112, permeate outlet 114, andconcentrate outlet 116. The flow chamber 110 is further defined by asidewall 115 to which the ultrasonic transducer 120 and the reflector130 are coupled. As seen here, the sidewall is shaped so that theultrasonic transducer and reflector are located on opposite sidesthereof.

Inlet 112 is located at a first end 106 of the flow chamber 110. Inparticular embodiments, the ingress of material through the inlet 112can be configured to occur toward the bottom end of the inlet 112, suchthat the inflow of fluid into the flow chamber 110 occurs closer to thebottom end of the flow chamber 110 than the top end thereof.

As depicted in FIG. 5 and FIG. 6, the inlet 112 is located along a firstside 107 of the device 100. The first side 107 of the device also housesthe reflector 130, while a second side 109 of the device, opposite thefirst side thereof, houses the ultrasonic transducer 120. The inlet 112could alternatively be located along the second side 109 of the device(e.g., on the same side as the ultrasonic transducer) or on another sideof the device.

In the embodiment depicted in FIG. 5, the permeate outlet 114 is locatedat a second end 108 of the flow chamber 100. The permeate outlet 114 isgenerally used to recover the first media and residual cells from theflow chamber 110. In comparison, the concentrate outlet 116 is locatedbetween the inlet 112 and the permeate outlet 114, below the ultrasonictransducer 120 and the reflector 130. The concentrate outlet 116 isgenerally configured to recover the cells from the flow chamber 110. Incertain embodiments, however, it may be desired to recover othermaterial (e.g., microcarriers) from the device, in which case themicrocarriers can be recovered by the concentrate outlet and the cellscan be recovered via the permeate outlet along with the media). As seenhere, the permeate outlet 114 is generally located above the ultrasonictransducer 120 and the reflector 130, while and the concentrate outlet116 is generally located below the ultrasonic transducer 120 and thereflector 130.

In the embodiment depicted in FIG. 5 and FIG. 6, the device 100 isvertically oriented, such that the first end 106 of the device is thebottom end thereof and the second end 108 of the device is the top endthereof. In this way, the cells surrounded by the first media isintroduced at the bottom end of the device 100 and flows verticallyupwards through the flow chamber from the inlet 112 toward the permeateoutlet 114.

As can be best seen in FIG. 6, the device 100 also includes a collector140. The collector 140 is located in the flow chamber 110 between theinlet 112 and the ultrasonic transducer 120 and the reflector 130. Thecollector 140 is located above the concentrate outlet 116 and, inparticular, is defined by angled walls 142 that lead to the concentrateoutlet 116. Put another way, the collector 140 leads into a common welldefined by angled walls 142 that taper downwards in cross-sectional area(i.e. larger area to smaller area) to a vertex at the bottom of thecollector, which is fluidically connected to and drains off one sideinto the concentrate outlet 116 via flowpath 119. In this way, themulti-dimensional acoustic standing wave can direct the concentratedcells to the collector 140 for collection and removal from the flowchamber 110 via the concentrate outlet 116. An annular plenum 117surrounds the collector 140, permitting the mixture of host fluid/cellsto flow from the inlet 112 around the collector 140 into the flowchamber 110.

In some embodiments, the collector leads to a collection container thatis filled with the second media. In this way, the second media is notflowed through the flow chamber of the device. Instead, as the cells aretrapped in the acoustic standing wave and form clusters that grow to acritical size and subsequently fall out of the multi-dimensionalacoustic standing wave, the cell clusters fall into the collector andare led to the collection container. The collection container can beseparable from the rest of the device.

As seen here, preferably, fluid flows through the device upwards. Thecells surrounded by the first media enters the device through inlet 112at a bottom end of the device and then makes a sharp turn to flowupwards. This change in direction desirably reduces turbulence,producing near plug flow upwards through the device. Flow continuesupwards through the annular plenum 117 and up into the flow chamber 110.There, the cells encounter the multi-dimensional acoustic standingwave(s), which traps the cells, as explained herein. Concentration ofthe cells occurs within the acoustic standing wave(s), which can alsocause coalescence, clumping, aggregation, agglomeration and/orclustering of the cells.

As the cells are concentrated, they eventually overcome the combinedeffect of the fluid flow drag forces and acoustic radiation force, andthey fall downwards into collector 140. They can then be flowed throughflowpath 119 and collected at concentrate outlet 116. A much highernumber of cells is obtained in a smaller volume (i.e., the target cellsare concentrated).

FIG. 7 is a graph showing the performance of the acoustophoretic deviceof FIG. 5. The device was operated at a fixed frequency of 2.234 MHz fora mixture having a feed cell density of about 1.5×10⁶ cells/mL. As canbe seen, the device achieved a permeate density reduction (PDR) ofgreater than 95% over about 35 minutes and a permeate cell density ofless than 0.10×10⁶ cells/mL over the same time period.

The piezoelectric transducer(s) of the acoustophoretic devices andsystems of the present disclosure can be single monolithic piezoelectricmaterials or can be made from an array of piezoelectric materials. Thepiezoelectric material can be a ceramic material, a crystal or apolycrystal, such as PZT-8 (lead zirconate titanate). FIG. 8 shows amonolithic, one-piece, single electrode piezoelectric crystal 200. Thepiezoelectric crystal has a substantially square shape, with a length203 and a width 205 that are substantially equal to each other (e.g.about one inch). The crystal 200 has an inner surface 202, and thecrystal also has an outer surface 204 on an opposite side of the crystalwhich is usually exposed to fluid flowing through the acoustophoreticdevice. The outer surface and the inner surface are relatively large inarea, and the crystal is relatively thin (e.g. about 0.040 inches for a2 MHz crystal).

FIG. 9 shows a piezoelectric crystal 200′ made from an array ofpiezoelectric materials. The inner surface 202 of this piezoelectriccrystal 200′ is divided into a piezoelectric array 206 with a pluralityof (i.e. at least two) piezoelectric elements 208. However, the array isstill a single crystal. The piezoelectric elements 208 are separatedfrom each other by one or more channels or kerfs 210 in the innersurface 202. The width of the channel (i.e. between piezoelectricelements) may be on the order of from about 0.001 inches to about 0.02inches. The depth of the channel can be from about 0.001 inches to about0.02 inches. In some instances, a potting material 212 (e.g., epoxy,Sil-Gel, and the like) can be inserted into the channels 210 between thepiezoelectric elements. The potting material 212 is non-conducting, actsas an insulator between adjacent piezoelectric elements 208, and alsoacts to hold the separate piezoelectric elements 208 together. Here, thearray 206 contains sixteen piezoelectric elements 208 (although anynumber of piezoelectric elements is possible), arranged in a rectangular4×4 configuration (square is a subset of rectangular). Each of thepiezoelectric elements 208 has substantially the same dimensions as eachother. The overall array 200′ has the same length 203 and width 205 asthe single crystal illustrated in FIG. 8.

FIG. 10 shows another embodiment of a transducer 200″. The transducer200″ is substantially similar to the transducer 200′ of FIG. 9, exceptthat the array 206 is formed from twenty-five piezoelectric elements 208in a 5×5 configuration. Again, the overall array 200″ has the samelength 203 and width 205 as the single crystal illustrated in FIG. 8.

Each piezoelectric element in the piezoelectric array of the presentdisclosure may have individual electrical attachments (e.g. electrodes),so that each piezoelectric element can be individually controlled forfrequency and power. These elements can share a common ground electrode.This configuration allows for not only the generation of amulti-dimensional acoustic standing wave, but also improved control ofthe acoustic standing wave. In this way, it is possible to driveindividual piezoelectric elements (or multiple, separate ultrasonictransducers) with arbitrary phasing and/or different or variablefrequencies and/or in various out-of-phase modes. For example, FIG. 11illustrates an exemplary 0-180-0-180 mode, though additional modes canbe employed as desired, such as a 0-180-180-0 mode. For example, for a5×5 array, additional modes can be employed as desired, such as a0-180-0-180-0 mode, a 0-0-180-0-0 mode, a 0-180-180-180-0 mode, or a0-90-180-90-0 mode. The array could also be driven, for example, suchthat a checkerboard pattern of phases is employed, such as is shown inFIG. 11. In summary, a single ultrasonic transducer that has beendivided into an ordered array can be operated such that some componentsof the array are out of phase with other components of the array.

The piezoelectric array can be formed from a monolithic piezoelectriccrystal by making cuts across one surface so as to divide the surface ofthe piezoelectric crystal into separate elements. The cutting of thesurface may be performed through the use of a saw, an end mill, or othermeans to remove material from the surface and leave discrete elements ofthe piezoelectric crystal between the channels/grooves that are thusformed.

As explained above, a potting material may be incorporated into thechannels/grooves between the elements to form a composite material. Forexample, the potting material can be a polymer, such as epoxy. Inparticular embodiments, the piezoelectric elements 208 are individuallyphysically isolated from each other. This structure can be obtained byfilling the channels 210 with the potting material, then cutting,sanding or grinding the outer surface 204 down to the channels. As aresult, the piezoelectric elements are joined to each other through thepotting material, and each element is an individual component of thearray. Put another way, each piezoelectric element is physicallyseparated from surrounding piezoelectric elements by the pottingmaterial. FIG. 12 is a cross-sectional view comparing these twoembodiments. On top, a crystal as illustrated in FIG. 9 is shown. Thecrystal is kerfed into four separate piezoelectric elements 208 on theinner surface 202, but the four elements share a common outer surface204. On the bottom, the four piezoelectric elements 208 are physicallyisolated from each other by potting material 212. No common surface isshared between the four elements.

FIG. 13 is a graph showing the performance of an acoustophoretic deviceaccording to the present disclosure having a 16-element piezoelectricarray. The piezoelectric array was operated at a fixed frequency of2.244 MHz for a mixture having a feed cell density of about 2.00×10⁶cells/mL. As can be seen, the device achieved a permeate densityreduction (PDR) of about 95% over about 60 minutes and a permeate celldensity of about 0.10×10⁶ cells/mL over the same time period.

The concentration efficiency of the acoustophoretic device was tested.First, a T-cell suspension having a cell density of 1×10⁶ cells/mL wasused. A feed volume of between about 500 and 1000 mL was used at a flowrate of 10-15 mL/minute. The results are graphically depicted in FIG.14. The device exhibited a concentration factor of between 10× and 20×,a 90% cell recovery, and a 77% washout efficiency (i.e., the amount ofthe first media that was displaced by the second media) over ten minutesof testing. A 10° C. temperature increase was observed.

A yeast mixture was then used to test the dependency of the percentdensity reduction (PDR) on concentration and flow rate. The results aregraphically depicted in FIG. 15. As seen here, the higher initial cellconcentrations generally resulted in a greater PDR. Additionally, thevaried flow rate (from 20 mL/min to 10 mL/min) did not have an observedeffect on the PDR.

The concentration efficiency of the acoustophoretic device was againtested with a higher cell density. A T-cell suspension having a celldensity of 5×106 cells/mL was used. A feed volume of 1000 mL was used ata flow rate of 10-15 mL/minute. The results are graphically depicted inFIG. 16. The device exhibited a concentration factor of better than 10×,a 90% cell recovery, and a 77% washout efficiency over one hour oftesting. A 10° C. temperature increase was again observed.

During testing, it was also discovered that active cooling of theultrasonic transducer led to greater throughput and efficiency and morepower. As such, a cooling unit was developed for actively cooling thetransducer. FIG. 17A illustrates an acoustophoretic device 7000containing a cooling unit, in a fully assembled condition. FIG. 17Billustrates the device 7000, with the various components in a partiallyexploded view. Referring now to FIG. 17B, the device includes anultrasonic transducer 7020 and a reflector 7050 on opposite walls of aflow chamber 7010. It is noted that the reflector 7050 may be made of atransparent material, such that the interior of the flow chamber 7010can be seen. The ultrasonic transducer is proximate a first wall of theflow chamber. The reflector is proximate a second wall of the flowchamber or can make up the second wall of the flow chamber. A coolingunit 7060 is located between the ultrasonic transducer 7020 and the flowchamber 7010. The cooling unit 7060 is thermally coupled to theultrasonic transducer 7020. In this figure, the cooling unit is in theform of a thermoelectric generator, which converts heat flux (i.e.temperature differences) into electrical energy using the Seebeckeffect, thus removing heat from the flow chamber. Put another way,electricity can be generated from undesired waste heat while operatingthe acoustophoretic device.

It is noted that the various inlets and outlets (e.g. fluid inlet,concentrate outlet, permeate outlet, recirculation outlet, bleed/harvestoutlet) of the flow chamber are not shown here. The cooling unit can beused to cool the ultrasonic transducer, which can be particularlyadvantageous when the device is to be run continuously with repeatedprocessing and recirculation for an extended period of time (e.g.,perfusion).

Alternatively, the cooling unit can also be used to cool the fluidrunning through the flow chamber 7010. For desired applications, thecell culture should be maintained around room temperature (−20° C.), andat most around 28° C. This is because when cells experience highertemperatures, their metabolic rates increase. Without a cooling unit,however, the temperature of the cell culture can rise as high as 34° C.

These components are modular and can be changed or switched outseparately from each other. Thus, when new revisions or modificationsare made to a given component, the component can be replaced while theremainder of the system stays the same.

The goal is to begin with a culture bag having a volume of about 1 liter(L) to about 2 L, with a density of about 1 million cells/m L, andconcentrate this bag to a volume of about 25 mL to about 30 mL, and thento wash the growth media or exchange the media within a time of aboutone hour (or less). Desirably, the system can be made of materials thatare stable when irradiated with gamma radiation.

The advantages of providing a cooling unit for the transducer can beseen in FIG. 18 and FIG. 19. FIG. 18 graphically shows the temperatureprofile of the acoustophoretic device without any active cooling (e.g.,without a cooling unit for the transducer). As seen in FIG. 18, thetemperature difference between the feed and the core (e.g., thetransducer) was 8.6° C. FIG. 19 graphically shows the temperatureprofile of the acoustophoretic device with active cooling (e.g., with acooling unit for the transducer). As seen in FIG. 19, through the use ofactive cooling the temperature difference between the feed and the corewas reduced to 6.1° C.

FIG. 20 illustrates a four-step process (with an optional fifth step)for concentrating, washing, and separating microcarriers from cells. Thefirst step in the process involves concentrating the microcarriers withattached cells in an acoustophoretic device, such as those describedherein. The microcarriers and attached cells can be introduced to theacoustophoretic device by receiving the microcarriers with attachedcells from a bioreactor. In the bioreactor, the microcarriers and cellsare suspended in a first media (e.g., growth serum or preservativematerial used to keep the cells viable in the bioreactor). Themicrocarriers with attached cells surrounded by the first media areconcentrated by the acoustic standing wave(s) generated in theacoustophoretic device. In a second step, the concentrated microcarrierswith attached cells are then washed with a second media to remove thefirst media (e.g., bioreactor growth serum or preservative material).The third step is to then introduce a third media containing an enzymeinto the acoustophoretic device to detach the cells from themicrocarriers through enzymatic action of the second media. Inparticular embodiments, trypsin is the enzyme used to enzymaticallydetach the cells from the microcarriers. The multi-dimensional acousticstanding wave can then be used to separate the cells from themicrocarriers. Usually, this is done by trapping the microcarriers inthe multi-dimensional acoustic standing wave, while the detached cellspass through with the third media. However, the cells can be trappedinstead, if desired. Finally, the separated cells may optionally beconcentrated and washed again, as desired.

After being concentrated and trapped/held in the multi-dimensionalacoustic standing wave, the microcarriers can coalesce, clump,aggregate, agglomerate, and/or cluster to a critical size at which pointthe microcarriers fall out of the acoustic standing wave due to enhancedgravitational settling. The microcarriers can fall into a collector ofthe acoustophoretic device located below the acoustic standing wave, tobe removed from the flow chamber.

During testing, steps one and two (i.e., concentration and washing) wereperformed using red and blue food dye to make colored fluid. Theconcentration mixture included SoloHill microcarriers in red fluid. Thewash mixture included blue fluid and was passed through the device threetimes. The concentrate was observed under a microscope, as shown in theleftmost image of FIG. 21. The concentration step was shown to have a99% efficiency. The remaining three images in FIG. 21 show microscopicimages after the first, second, and third wash passes, respectively. Asseen from left to right in FIG. 21, the first media (dyed red) isprogressively washed out by a second media (dyed blue) over a series ofwash passes. The light absorbance data is shown in the table below.

Light Absorbance Sample Red (510 nm) Blue (630 nm) Feed 0.138 0.041 WashPass 1 0.080 0.066 Wash Pass 2 0.063 0.080 Wash Pass 3 0.054 0.084

The decrease in red light absorbance and increase in blue lightabsorbance evidences the feasibility of the washing steps.

FIG. 22 shows microscopic images of the microcarriers and attached cellsduring the concentration and washing steps. In particular, the leftmostimage in the top row shows the microcarriers and attached cells in thefeed, prior to introduction into the acoustophoretic device. Therightmost image in the top row shows the microcarriers and attachedcells in the permeate, after concentration in the acoustophoreticdevice. The bottom row of images show the microcarriers and attachedcells in the device during the washing step, namely during the first,second, and third wash passes, from left to right.

FIG. 23 shows the concentration of T-cells after being separated in theacoustophoretic device. The top row of images show the T-cells beforeacoustophoresis with a concentration of 1.14±0.03×10⁶ cells/mL. Thebottom row of images show the T-cells after acoustophoresis with aconcentration of 1.13±0.02×10⁶ cells/mL. The comparable concentrationsevidence that substantially all of the cells pass through theacoustophoretic device, as the concentration was substantially unchangedby acoustophoresis.

FIG. 24 shows the presence of SoloHill microcarriers and T-Cells in theacoustophoretic device under 4× magnification. The top row of imagesshow the microcarriers and cells in the feed before acoustophoresis. Thebottom row of images show the microcarriers and cells in the permeateafter the cells have been separated out by acoustophoresis. Thedifference in the number of microcarriers with the application ofacoustophoresis evidences the feasibility of using the device fortrapping the microcarriers in the device and separating the cellstherefrom. The feasibility of this technique and the results are furtherevidenced by the images in FIG. 25, which show microscopic images of themicrocarriers and cells in the feed (top row of images) and permeate(bottom row of images) after concentration and the first, second, andthird washes, from left to right.

The testing of the acoustophoretic concentrating, washing, andseparating process showed that the process is appropriate for celltherapy and microcarrier applications. The concentrate and wash stepswere performed with a resulting efficiency of greater than 99%, and theseparating step e.g., separating the cells from the microcarriers, wasperformed with greater than 98% efficiency.

FIGS. 26-28 illustrate another example embodiment of an acoustophoreticsystem/process 2800 including a disposable acoustophoretic device 2810with solenoid pinch valves that control the flow of fluid therethrough.Starting from the left-hand side of FIG. 26, the system includes a feedtank 2820, a wash tank 2830, and an air intake 2805. The air intake 2805runs through an air intake valve 2804. Feed line 2821 runs from the feedtank 2820. The air intake and the feed line 2821 are joined together bya Y-connector into common feed line 2811, which runs into feed selectorvalve 2801. A wash line 2831 runs from the wash tank 2830, and also runsinto feed selector valve 2801. Feed selector valve 2801 permits only oneline to be open at a given time (valves 2802, 2803 also operate in thismanner). Wash line 2831 and feed line 2811 are joined together by aY-connector downstream of the feed selector valve 2801 into input line2812. Input line 2812 passes through pump 2806 to inflow selector valve2802, which is downstream of the feed selector valve 2801 and upstreamof the acoustophoretic device 2810. The inflow selector valve 2802selectively controls the inflow of feed or wash into the acoustophoreticdevice 2810 through either feed port 2602 or wash/drain port 2604. Afeed line 2813 runs from the inflow selector valve 2802 to feed port2602. A wash line 2814 runs from the inflow selector valve 2802 tocommon line 2815 and into wash/drain port 2604.

On the right-hand side of FIG. 26, an outflow selector valve 2803 islocated downstream of the acoustophoretic device 2810 and controls theoutflow of fluid therefrom. A waste line 2816 runs from waste port 2608through outflow selector valve 2803 and subsequently to waste tank 2850.The common line 2815 runs into drain line 2817, which then passesthrough outflow selector valve 2803 and subsequently to concentrate tank2840. These tanks 2840, 2850 can be, for example, collection bags. Theoutflow selector 2803 thereby selectively controls the flow of fluid tothe concentrate tank and waste tank.

The use of collection bags at the ends of the concentrate and wastelines advantageously creates an enclosed primary environment withinwhich concentration, washing, and/or separation of cells and cellularmaterials can occur, which helps to prevent the cells/cellculture/cellular material from being exposed to possible intrusions,pathogens, or outside cellular influences that would be harmful.

FIG. 26 also illustrates the flow path of the feed material through thesystem. In this example embodiment, feed selector valve 2801 is operatedwith the bottom open (and top closed), so that the feed from feed tank2820 flows through. Inflow selector valve 2802 is operated with the topopen (and bottom closed), so that the feed material enters theacoustophoretic device 2810 via feed port 2602. The outflow selectorvalve 2803 is also operated with the top open (and bottom closed) sothat the fluid/first media of the feed material flows through to wastetank 2850. The targeted particles in the feed material (e.g.,microcarriers or cells) are trapped in the acoustophoretic device 2810by action of a multi-dimensional acoustic standing wave(s), as explainedin detail herein.

FIG. 27 illustrates the flow path of the wash material through thesystem. Feed selector valve 2801 is operated with the top open (andbottom closed), so that the wash material from wash tank 2830 flowsthrough. The inflow selector valve 2802 is operated with the bottom open(and top closed) and the outflow selector valve 2803 is operated withthe top open (and bottom closed). As a result, the wash material entersthe acoustophoretic device 2810 via wash/drain port 2604, which operatesas a wash inlet. Note that the closed outflow selector valve 2803prevents the wash material from entering concentrate tank 2840. The washmaterial can then pass through the acoustophoretic device 2810 andremove the first media (e.g., bioreactor serum or preservativematerial). The wash material then exits via waste port 2608 and flows towaste tank 2850. The target particles remain trapped in theacoustophoretic device 2810.

FIG. 28 illustrates the draining of the system (e.g., the collection ofthe target particles). Air intake valve 2804 is opened. The feedselector valve 2801 is operated with the bottom open (and top closed),and the inflow selector valve 2802 is operated with the top open (andbottom closed), so that air enters the acoustophoretic device 2810 viafeed port 2602. The air generally aids in dislodging the clusters oftarget particles from the acoustophoretic device 2810. The outflowselector valve 2803 is operated with the bottom open (and top closed).The target particles flow out of wash/drain port 2604 through commonline 2815, through drain line 2817 and subsequently to concentrate tank2840.

Concentrating and washing cell culture is useful for producingbiological products for industrial use. The systems of the presentdisclosure can be continuously improved and scaled up for handling oflarger volumes.

In some examples, the acoustophoretic devices of the present disclosuremay have a concentrated volume ranging from about 25 mL to about 75 mL.The devices may have a total cell capacity of about 4 billion to about40 billion cells, or from about 4 billion to about 8 billion cells, orfrom about 20 billion to about 40 billion cells, or from about 16billion to about 35 billion cells. The fluids entering and exiting theacoustophoretic devices may have cell densities from about 160 millioncells/mL to about 670 million cells/mL, or from about 160 millioncells/mL to about 320 million cells/mL, or from about 260 millioncells/mL to about 535 million cells/mL, or from about 305 millioncells/mL to about 670 million cells/mL, or from about 0.5 millioncells/mL to about 5 million cells/m L.

The following examples are provided to illustrate the devices andprocesses of the present disclosure. The examples are merelyillustrative and are not intended to limit the disclosure to thematerials, conditions, or process parameters set forth therein.

EXAMPLES

The ability of an acoustophoretic system of the present disclosure toconcentrate Jurkat T-cells was tested. Jurkat T-cells have a diameter of11 micrometers (μm) to 14 μm. An acoustophoretic device was used, and aBeckman Coulter Vi-CELL X was used at various test conditions to measurethe cell density reduction.

In the first trial A, the T-cells were concentrated, and the celldensity of the permeate was measured. The dotted line indicates the feedcell density. Desirably, the cell density in the permeate is as low aspossible, indicating that the cells are retained in the concentrate. Thegraph in FIG. 29 shows the results of trial A over time. The resultsshow very low cell densities in the permeate, between 0.0 and 0.2million cells/mL, showing that most of the cells are in the concentrate.The results also show a high permeate reduction percentage, between 80%and 99%.

In the second trial B, the T-cells were concentrated, and the celldensity of the permeate was measured. The dotted line indicates the feedcell density. FIG. 30 shows the results over time. The results show goodperformance, with the permeate cell density being below 0.1 millioncells/mL after minute 1, and greater than 95% permeate reduction afterminute 2.

In the third trial C, the T-cells were concentrated and washed. Theconcentrating occurred for the first 18 minutes, and washing wassubsequently performed. FIG. 31 shows the results over time. The dottedline indicates the feed cell density. The solid vertical lines indicatewhen concentrated system volumes were processed (three total volumeswere processed). Note that this graph includes data on the concentrateand the permeate (not just the permeate). All of the cells obtained fromconcentration were maintained through washing, e.g., concentrated cellswere not lost due to the addition of the washing process. The tablebelow provides additional information on these three trials. Retentionand recovery rates of greater than 90% were obtainable for JurkatT-cells.

Feed Feed Concen- Process Volume Density Concentrate Cell tration TimeTrial (mL) (cells/mL) Volume Recovery Factor (min) A 997 0.98 × 10⁶ 21mL 91% 47X 33 B 1004 0.85 × 10⁶ 21 mL 95% 48X 33 C 555 4.08 × 10⁶ 20 mL92% 28X 31

The liquid volumes used to completely wash the concentrated cells weretracked. Tracking the liquid volumes can be useful in applications suchas, for example, removing electroporation buffer from a cell cultureprior to transduction or transfection of the cell culture.

A blue wash media and a red growth media were used. A Molecular DevicesSpectraMax spectrophotometer was used to measure the two differentwavelengths of these two media to identify a complete flush/washing outof the old growth media from the system. Three samples were measured:100% wash media (100 W-0 G), 50% wash media and 50% growth media (50W-50 G), and 100% growth media (0 W-100 G). Three samples of the actualprocess were then tested (1 Volume, 2 Volume, 3 Volume). As seen in thespectrophotometer results shown in FIG. 32, the second and third volumesfall on top of the 100% wash media curve (100 W-0 G), indicating thatall of the growth media has been washed from the concentrated cellsafter 2 or 3 volumes have been used for washing.

The present disclosure has been described with reference to exemplaryembodiments. Modifications and alterations may occur to others uponreading and understanding the preceding detailed description. It isintended that the present disclosure be construed as including all suchmodifications and alterations insofar as they come within the scope ofthe appended claims or the equivalents thereof.

1. A method exchanging media, the method comprising: providing aninitial mixture of a first media and particles to a chamber of anacoustophoretic device, the acoustophoretic device including at leastone ultrasonic transducer that includes a piezoelectric material;driving the at least one ultrasonic transducer to create an acousticwave an acoustic region in the chamber; providing the initial mixture tothe acoustic region, such that at least a portion of the particles aretrapped and held against fluid flow in the acoustic region; formingclusters of the trapped particles to grow in size in the acousticregion; and flowing a second media to the chamber while the particlesand clusters are retained in the chamber to cause the first media toflow out of the chamber; wherein an input rate of one or more of theinitial mixture or the second media to the chamber is in a range of fromabout 10 ml/min to about 15 ml/min.
 2. The method of claim 1, whereinthe second media is a biocompatible wash or a buffer solution.
 3. Themethod of claim 1, wherein the particles are cells.
 4. The method ofclaim 1, wherein the particles are microcarrier/cell complexes.
 5. Themethod of claim 1, wherein the initial mixture has a density of about0.5 million particles/ml to about 5 million particles/ml.
 6. The methodof claim 1, further comprising concentrating the particles in theinitial mixture.
 7. The method of claim 6, further comprisingconcentrating the particles to a concentrate volume that is about 25 toabout 50 times less than a volume of the initial mixture.
 8. The methodof claim 7, further comprising concentrating the particles in theinitial mixture to a concentrated particle density of about 25 to about50 times greater than a particle density of the initial mixture.
 9. Themethod of claim 1, wherein a cell density of the first media output fromthe chamber is about 0.0 to about 0.5 million cells/ml.
 10. The methodof claim 9, wherein the first media output is from a concentrate processand a wash process.
 11. The method of claim 1, further comprisingconducting a spectrophotometer process on the chamber to determine washefficacy.
 12. A method of recovering cells from a cell culture,comprising: feeding an initial mixture of the cell culture to a flowchamber of an acoustophoretic device, the acoustophoretic deviceincluding at least one ultrasonic transducer that includes apiezoelectric material that is configured to be driven to generate amulti-dimensional acoustic wave in the flow chamber; and driving the atleast one ultrasonic transducer to generate a multi-dimensional acousticwave in an acoustic region in the flow chamber; providing the initialmixture to the acoustic region; and retaining the cells from the initialmixture in the acoustic region to form clusters of the cells to grow insize in the acoustic region; wherein an input rate of the initialmixture to the flow chamber is in a range of from about 10 ml/min toabout 15 ml/min.
 13. The method of claim 12, wherein the cell density ofthe concentrated cells is about 25 to about 50 times greater than thecell density of the initial mixture.
 14. The method of claim 12, whereina volume of the concentrated cells is 25 to about 50 times less than avolume of the initial mixture.
 15. The method of claim 12, wherein theconcentrated cells are obtained in about 35 minutes or less.
 16. Themethod of claim 12, further comprising washing the concentrated cells,wherein a cell density of a wash output of the flow chamber is about 0.0to about 0.5 million cells/ml.
 17. An acoustophoretic device,comprising: a flow chamber with a first outlet; at least one ultrasonictransducer coupled to the flow chamber and including a piezoelectricmaterial that is adapted to be driven to generate an acoustic wave in anacoustic region of the flow chamber, such that particles are trapped toform particle clusters in the acoustic region that can grow in size whena fluid and particle mixture is provided to the acoustic region; adiminished particle concentration region adjacent the acoustic regionand in fluid communication with the first outlet, the diminishedparticle concentration region being interposed between the acousticregion and the first outlet; and a fluid input region on an oppositeside of the acoustic region from the first outlet that permits fluidflow into the acoustic region, such that an input fluid flows throughthe acoustic region while the particles and particle clusters remaintrapped in the acoustic region; wherein the dimensions of the flowchamber and the first outlet are sized to accommodate a flow rate in arange of from about 10 ml/min to about 15 ml/min.
 18. Theacoustophoretic device of claim 17, wherein the flow chamber can containa cell capacity of about 4 billion to about 40 billion cells.